Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolKnodZ as well as noeC and/or downstream genes

Abstract

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild-type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D-arabinosyl and/or an L-fucosyl branch. Nod factors produced by Tn5-insertion mutants in nodZ, noeC, and the separate nolK locus, were analysed by thin-layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK. Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP-fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ. In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP-fucose.

Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

“…Attempts in our laboratory to purify and sequence the responsible enzymes from membrane preparations have been unsuccessful. The necessary breakthrough came when those of us working on Azorhizobium, one of the few groups of organisms outside Actinomycetales to synthesize Darabinofuranose, were able to identify a D-arabinose operon by gene knock-out studies 3 (11). These studies helped by allowing the precise pathway from pRpp to decaprenylphosphoryl-Darabinose to be determined 2 and, as shown here, to identify and express the gene encoding the first enzyme in the pathway, a new phosphoribosyl transferase that transfers ribose-5-phosphate to decaprenyl phosphate.…”

“…Decaprenyl-phosphate 5-Phosphoribosyltransferase-Earlier studies by some of us revealed that A. caulinodans D-arabinofuranosylated the nod factor produced by this nitrogen-fixing symbiotic organism (11,13). Furthermore, a gene cluster responsible for this arabino- sylation was identified by knock-out experiments.…”

“…The first reaction in the formation of decaprenylphosphoryl-Darabinose is the transfer of ribose-5-phosphate from pRpp to decaprenylphosphate 2 (10) to form decaprenylphosphoryl-5-phosphoribose (DPPR). Genetic analysis of a gene cluster found in Azorhizobium caulinodans responsible for D-arabinosylation of nod factor 3 (11) revealed four open reading frames; three of these have homology to M. tuberculosis genes. Rv3806c, which shows homology to the noeC gene of A. caulinodans, was hypothesized to be the 5-phospho-␣-D-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase gene because of its transmembrane domains and its homology to prenyl transferases.…”

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island

“…HPLC-MS analysis (SI Appendix, Fig. S7B) showed that this fraction contained naringenin, a common flavonoid compound, which has been shown to induce nod gene expression in A. caulinodans (18). We thus selected naringenin to examine the role of plant signals in HGT.…”

Identification of amino acids and domains required for catalytic activity of DPPR synthase, a cell wall biosynthetic enzyme of Mycobacterium tuberculosis

“…To search for homologues of the DPPR synthase of M. tuberculosis, the amino acid sequence of Rv3806c was used as a query in a homology search of the TIGR database (http://tigrblast.tigr.org/cmr-blast/). The forward primers used to amplify the DPPR synthase genes of M. tuberculosis (Rv3806c), Mycobacterium smegmatis (MSMEG_6401), Corynebacterium glutamicum (cg3189) and Azorhizobium caulinodans (noeC) (Mergaert et al, 1996) included an NdeI restriction site (underlined) while the reverse primers had a BamHI restriction site (underlined) downstream of the stop codon. The primer sequences were: M. tuberculosis (Fw) 59-CATATGAGTGAAGATGTGGTGAC-TCAACC-39, (Rev) 59-GGATCCCTAGCCGAAGGCAACAGCGGC-39; M. smegmatis (Fw) 59-CATATGGATGCCACCCACATGAGTG-39, (Rev) 59-GGATCCTCAGCTGAAATAGATGGCCGC-39; C. glutamicum (Fw) 59-CATATGAGCGAACACGCCGCTGAAC-39, (Rev) 59-GGATCCTCAAAACATCGGCATGATGTAC-39; A. caulinodans (Fw) 59-CATATGTGGAATAAGGAATGGCCCG-39, (Rev) 59-GGATCC-TCAACCAATCGATTGCGACCG-39.…”